Use of multivalent inorganic cations in the electrochemical treatment of nucletic acid containing solutions

ABSTRACT

A process is described for denaturing native double-stranded nucleic acid material into its individual strands in an electrochemical cell. The process is an electrical treatment of the nucleic acid with a voltage applied to the nucleic acid material by an electrode. The process employs a promoter which is an inorganic multivalent cation such as magnesium ions to speed denaturation. The process may be used in the detection of nucleic acid by hybridrising with a labelled probe or in the amplification of DNA by a polymerase chain reaction or ligase chain reaction.

This invention relates to processes for the treatment of nucleic acidmaterial in order to effect a complete or partial change from doublestranded form to single stranded form and to processes of amplifying ordetecting nucleic acids involving such denaturation processes.

BACKGROUND OF THE INVENTION

Double stranded DNA (deoxyribonucleic acid) and DNA/RNA (ribonucleicacid) and RNA/RNA complexes in the familiar double helical configurationare stable molecules that, in vitro, require aggressive conditions toseparate the complementary strands of the nucleic acid. Known methodsthat are commonly employed for strand separation require the use of hightemperatures of at least 60° celsius and often 100° celsius for extendedperiods of ten minutes or more or use an alkaline pH of 11 or higher.Other methods include the use of helicase enzymes such as Rep protein ofE. coli that can catalyse the unwinding of the DNA in an unknown way, orbinding proteins such as 32-protein of E.coli phage T4 that act tostabilise the single stranded form of DNA. The denatured single strandedDNA produced by the known processes of heat or alkali is used commonlyfor hybridisation studies or is subjected to amplification cycles.

U.S. Pat. No. 4,683,202 (Kary B Mullis et al, assigned to CetusCorporation) discloses a process for amplifying and detecting a targetnucleic acid sequence contained in a nucleic acid or mixture thereof byseparating the complementary strands of the nucleic acid, hybridisingwith specific oligonucleotide primers, extending the primers with apolymerase to form complementary primer extension products and thenusing those extension products for the further synthesis of the desirednucleic acid sequence by allowing hybridisation with the specificoligonucleotides primers to take place again. The process can be carriedout repetitively to generate large quantities of the required nucleicacid sequence from even a single molecule of the starting material.Separation of the complementary strands of the nucleic acid is achievedpreferably by thermal denaturation in successive cycles, since only thethermal process offers simple reversibility of the denaturation processto reform the double stranded nucleic acid, in order to continue theamplification cycle. However the need for thermal cycling of thereaction mixture limits the speed at which the multiplication processcan be carried out owing to the slowness of typical heating and coolingsystems. It also requires the use of special heat resistant polymeraseenzymes from thermophilic organisms for the primer extension step if thecontinuous addition of heat labile enzyme is to be avoided. It limitsthe design of new diagnostic formats that use the amplification processbecause heat is difficult to apply in selective regions of a diagnosticdevice and it also can be destructive to the structure of the DNA itselfbecause the phosphodiester bonds may be broken at high temperaturesleading to a collection of broken single strands. It is generallybelieved that the thermophilic polymerases in use today have a lowerfidelity ie. make more errors in copying DNA than do enzymes frommesophiles. It is also the case that thermophilic enzymes such as TAQpolymerase have a lower turnover number than heat labile enzymes such asthe Klenow polymerase from E.coli. In addition, the need to heat to hightemperatures, usually 90° celsius or higher to denature the nucleic acidleads to complications when small volumes are used as the evaporation ofthe liquid is difficult to control. These limitations have so far placedsome restrictions on the use of the Mullis et al process in applicationsrequiring very low reagent volumes to provide reagent economy, inapplications where the greatest accuracy of copy is required such as inthe Human Genome sequencing project and in the routine diagnosticsindustry where reagent economy, the design of the assay format and thespeed of the DNA denaturation/renaturation process are important.

Denaturation/renaturation cycles are also required in order to performthe so-called ligase chain reaction described in EP-A-0320308 in whichamplification is obtained by ligation of primers hybridised to templatesequences rather than by extending them.

It is known that DNA has electrochemical properties. For example, N. L.Palacek (in "Electrochemical Behaviour of Biological Macromolecules",Bioelectrochemistry and Bioenergetics, 15, (1986), 275-295) disclosesthe electrochemical reduction of adenine and cytosine in thermallydenatured single stranded DNA at about -(minus) 1.5 V on the surface ofa mercury electrode. This reduction process also requires a priorprotonation and therefore takes place at a pH below 7.0. The primaryreduction sites of adenine and cytosine form part of the hydrogen bondsin the Watson-Crick base pairs. Palacek was unable to demonstratereduction of adenine and cytosine in intact, native double stranded DNAat the mercury electrode. Palacek has further demonstrated that to avery limited extent the DNA double helix is opened on the surface of themercury electrode at a narrow range of potentials centred at -(minus)1.2V in a slow process involving an appreciable part of the DNA molecule.This change in the helical structure of the DNA is thought to be due toprolonged interaction with the electrode charged to certain potentialsand is not thought to be a process involving electron transfer to theDNA. No accumulation of single stranded DNA in the working solution wasobtained and no practical utility for the phenomenon was suggested.Palacek also reports that the guanine residues in DNA can be reduced at-(minus)1.8 V to dihydroguanine which can be oxidised back to guanine ataround -(minus)0.3 V. The reducible guanine double bond is not part ofthe hydrogen bonds in the Watson-Crick base pairs and thiselectrochemical process involving guanine does not affect the structureof the DNA double helix.

In an earlier paper F. Jelen and E. Palacek (in "NucleotideSequence-Dependent Opening of Double-Stranded DNA at an ElectricallyCharged Surface", Gen. Physiol. Biophys., (1985), 4, pp 219-237),describe in more detail the opening of the DNA double helix on prolongedcontact of the DNA molecules with the surface of a mercury electrode.The mechanism of opening of the helix is postulated to be anchoring ofthe polynucleotide chain via the hydrophobic bases to the electrodesurface after which the negatively charged phosphate residues of the DNAare strongly repelled from the electrode surface at an applied potentialclose to -(minus)1.2 V, the strand separation being brought about as aresult of the electric field provided by the cathode. There is nodisclosure of separating the strands of the DNA double helix while theDNA is in solution (rather than adsorbed onto the electrode) and thereis no disclosure of useful amounts of single strand DNA in solution.Furthermore, there is no disclosure that the nucleotide base sequence ofthe DNA on the electrode is accessible from solution. The basesthemselves are tightly bound to the mercury surface. A mercury electrodeis a complex system and the electrode can only be operated in theresearch laboratory with trained technical staff.

H W Nurnberg ("Applications of Advanced Voltammetric Methods inElectrochemistry" in "Bioelectrochemistry", Plenum Inc (New York), 1983,pp. 183-225) discloses partial helix opening of adsorbed regions ofnative DNA to a mercury electrode surface to form a so-called ladderstructure. However, the DNA is effectively inseparably bound to oradsorbed onto the electrode surface. In this condition, we believe thedenatured DNA to be of no use for any subsequent process ofamplification or analysis. To be of any use, the denatured DNA must beaccessible to subsequent processes and this is conveniently achieved ifthe single stranded DNA is available in free solution or is associatedwith the electrode in some way but remains accessible to furtherprocesses. Nurnberg has not demonstrated the ability of the mercuryelectrode to provide useful quantities of single stranded DNA.

V. Brabec and K. Niki ("Raman scattering from nucleic acids adsorbed ata silver electrode" in Biophysical Chemistry (1985), 23, pp 63-70) haveprovided a useful summary of the differing views from several workers onDNA denaturation at the surface of both mercury and graphite electrodescharged to negative potentials. There has emerged a consensus amongstthe research workers in this field that the denaturation process onlytakes place in DNA that is strongly adsorbed to the electrode surfaceand only over prolonged periods of treatment with the appropriatenegative voltage, a positive voltage having no effect on the doublehelix.

Brabec and Palacek (J. Electroanal. Chem., 88 (1978) 373-385) disclosethat sonicated DNA damaged by gamma radiation is transiently partiallydenatured on the surface of a mercury pool electrode, the process beingdetectable by reacting the single stranded products with formaldehyde soas to accumulate methylated DNA products in solution. Intact DNA did notshow any observable denaturation.

Our Application No. PCT/GB91/01563 discloses a process for denaturingdouble-stranded nucleic acid which comprises operating on solutioncontaining nucleic acid with an electrode under conditions such as toconvert a substantial portion of said nucleic acid to a wholly orpartially single stranded form.

This process was based on a finding that it is possible to produce thedenaturation of undamaged (i.e. non-irradiated) DNA at ambienttemperature by applying a suitable voltage to a solution in which theDNA is present under suitable conditions.

The mechanism for the process has not yet been fully elucidated. Webelieve that the process is one in which the electric field at theelectrode surface which produces the denaturation of the double helix.

In polymerase chain reaction processes, it has been shown that thedenatured DNA produced by the denaturing process is immediately in asuitable state for primer hybridisation and extension. On a largerscale, it has been found that samples of denatured DNA produced eitherby a negative voltage electrode or thermal denaturation can be caused orencouraged to reanneal by incubation at a higher temperature or by theuse of a positive voltage.

Although the process of Application No. PCT/GB91/01563 can take place ina solution containing only the electrode and the nucleic acid dissolvedin water containing a suitable buffer, the process can be facilitated bythe presence in the solution containing the nucleic acid of a promotercompound. Methyl viologen or a salt thereof was disclosed as thepreferred promoter compound.

It is believed that the positively charged viologen molecules interactbetween the negatively charged DNA and the negatively charged cathode toreduce electrostatic repulsion therebetween and hence to promote theapproach of the DNA to the electrode surface where the electrical fieldis at its strongest. Accordingly, we expressed a preference inApplication No. PCT/GB91/01563 to employ as promoters compounds havingspaced positively charged centres, e.g. bipolar positively chargedcompounds. Preferably, the spacing between the positively chargedcentres was to be similar to that in viologens.

SUMMARY OF THE INVENTION

We have now discovered that multivalent inorganic cations, preferablyMg²⁺, can also act as promoters in such a system with approximately thesame efficacy as methyl viologen.

It is thought that large cations such as Mg²⁺ are able to act as abridge between a negative electrode and negatively charged regions ofthe double-stranded nucleic acid.

Accordingly, the present invention provides a process for denaturingdouble-stranded nucleic acid which comprises operating on solutioncontaining said nucleic acid with an electrode under condition such asto convert a substantial proportion of said nucleic acid to a wholly orpartially single stranded form wherein the solution contains aneffective concentration of a multivalent inorganic cation acting as apromoter which assists said denaturation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The cations used as the promoter may include inorganic cations complexedwith inorganic or organic ligands, e.g. Pt(NH₃)₆ ⁴⁺ and Cr(NH₃)₆ ²⁺ butthe preferred cation is Mg²⁺. Mixtures of promoter cations may beemployed.

The concentration of said promoter cation is preferably from 1 mM to 250mM, more preferably from 70 mM, e.g. about 100 mM.

Preferably, according to the invention, the single stranded nucleic acidproduced is free from the electrode, e.g. in solution. However, thenucleic acid may be immobilised on the electrode in double or singlestranded form prior to the application of the electric potential, e.g.attached by the end or a small portion intermediate the ends of thenucleic acid chain, so as to leave substantial segments of the nucleicacid molecules freely pendant from the electrode surface before andafter denaturation.

In addition to said electrode and a counter-electrode, a referenceelectrode may be contacted with said solution and a voltage may beapplied between said electrode and said counter-electrode so as toachieve a desired controlled voltage between said electrode and saidreference electrode. The electrodes may be connected by a potentiostatcircuit as is known in the electrochemical art.

Preferably, a potential of from -0.5 to -1.5 V is applied to saidworking electrode with respect to said reference electrode, morepreferably from -0.8 to -1.1 V, e.g. about -1.0 V.

Working electrode voltages are given throughout as if measured or asactually measured relative to a calomel reference electrode (BDH No.309.1030.02).

The ionic strength of said solution is preferably no more than 250 mM,more preferably no more than 100 mM. As it has been found that the rateof denaturation increases as the ionic strength is decreased, the saidionic strength is still more preferably no more than 50 mM, e.g. no morethan 25 mM or even no more than 5 mM. Generally, the lower the ionicstrength, the more rapid is the denaturation. However, in calculatingionic strength for these purposes it may be appropriate to ignore onecontribution to ionic strength of any component which acts as a promoteras described above.

The process may be carried out in an electrochemical cell of the typedescribed by C. J. Stanley, M. Cardosi and A. P. F Turner "AmperometricEnzyme Amplified Immunoassays" J. Immunol. Meth (1988) 112, 153-161 inwhich there is a working electrode, a counter electrode and optionally areference electrode. The working electrode at or by which the denaturingnucleic acid is effected may be of any convenient material e.g. a noblemetal such as gold or platinum, or a glassy carbon electrode.

The electrode may be a so called "modified electrode" in which thedenaturing is promoted by a compound coated onto, or adsorbed onto, orincorporated into the structure of the electrode which is otherwise ofan inert but conducting material. In an alternative electrochemical cellconfiguration the working, counter and reference electrodes may beformed on a single surface e.g. a flat surface by any printing methodsuch as thick film screen printing, ink jet printing, or by using aphoto-resist followed by etching. It is also possible that the counterand reference electrodes can be combined on the flat surface leading toa two electrode configuration. Alternatively the electrodes may beformed on the inside surface of a well which is adapted to hold liquidsuch a well could be the well known 96 well or Microtitre plate, it mayalso be a test tube or other vessel. Electrode arrays in Microtitreplates or other moulded or thermoformed plastic materials may beprovided for multiple nucleic acid denaturation experiments.

The strand separation may be carried out in an aqueous medium or in amixture of water with an organic solvent such as dimethylformamide. Theuse of polar solvents other than water or non-polar solvents is alsoacceptable but is not preferred. The process may be carried out atambient temperatures or if desired temperatures up to adjacent thepre-melting temperature of the nucleic acid. The process may be carriedout at pH's of from 3 to 10 conveniently about 7. Generally, more rapiddenaturation is obtained at lower pH. For some purposes therefore a pHsomewhat below neutral, e.g about pH 5.5 may be preferred. The nucleicacid may be dissolved in an aqueous solution containing a buffer whosenature and ionic strength are such as not to interfere with the strandseparation process.

The denaturing process according to the invention may be incorporated asa step in a number of more complex processes, e.g. procedures involvingthe analysis and or the amplification of nucleic acid. Some examples ofsuch applications are described below.

The invention includes a process for detecting the presence or absenceof a predetermined nucleic acid sequence in a sample which comprises:denaturing a sample double-stranded nucleic acid by means of a voltageapplied to the sample in a solution by means of an electrode;hybridising the denatured nucleic acid with an oligonucleotide probe forthe sequence; and determining whether the said hybridisation hasoccurred, wherein during denaturing the solution contains an effectiveconcentration of a multivalent inorganic cation acting as a promoterwhich assists said denaturation.

Thus, the invented process has application in DNA and RNA hybridisationwhere a specific gene sequence is to be identified e.g. specific to aparticular organism or specific to a particular hereditary disease ofwhich sickle cell anaemia is an example. To detect a specific sequenceit is first necessary to prepare a sample of DNA, preferably of purifiedDNA, means for which are known, which is in native double stranded form.It is then necessary to convert the double stranded DNA to singlestranded form before a hybridisation step with a labelled nucleotideprobe which has a complementary sequence to the DNA sample can takeplace. The denaturation process of the invention can be used for thispurpose in a preferred manner by carrying out the following steps:

denaturing a sample of DNA by applying a voltage by means of anelectrode to the sample DNA with a said promoter in solution;

hybridising the denatured DNA with a directly labelled or indirectlylabelled nucleotide probe complementary to the sequence of interest; and

determining whether the hybridisation has occurred, which determinationmay be by detecting the presence of the probe, the probe being directlyradio-labelled, fluorescent labelled, chemiluminescent labelled orenzyme-labelled or being an indirectly labelled probe which carriesbiotin for example to which a labelled avidin or avidin type moleculecan be bound later.

In a typical DNA probe assay it is customary to immobilise the sampleDNA to a membrane surface which may be composed of neutral or chargednylon or nitrocellulose. The immobilisation is achieved by chargeinteractions or by baking the membrane containing DNA in an oven. Thesample DNA can be heated to high temperature to ensure conversion tosingle stranded form before binding to the membrane or it can be treatedwith alkali once on the membrane to ensure conversion to the singlestranded form. The disadvantages of the present methods are:

heating to high temperatures to create single stranded DNA can causedamage to the sample DNA itself.

the use of alkali requires an additional step of neutralisation beforehybridisation with the labelled probe can take place.

One improved method for carrying out DNA probe hybridisation assays isthe so called "sandwich" technique where a specific oligonucleotide isimmobilised on a surface. The surface having the specificoligonucleotide thereon is then hybridised with a solution containingthe target DNA in a single-stranded form, after which a second labelledoligonucleotide is then added which also hybridises to the target DNA.The surface is then washed to remove unbound labelled oligonucleotide,after which any label which has become bound to target DNA on thesurface can be detected later.

This procedure can be simplified by using the denaturing process of theinvention to denature the double-stranded DNA into the requiredsingle-stranded DNA. The working electrode, counter electrode andoptionally a reference electrode and/or the promoter can be incorporatedinto a test tube or a well in which the DNA probe assay is to be carriedout. The DNA sample, promoter if not already present and oligonucleotideprobes can then be added and the voltage applied to denature the DNA.The resulting single-stranded DNA is hybridised with the specificoligonucleotide immobilised on the surface after which the remainingstages of a sandwich assay are carried out. All the above steps can takeplace without a need for high temperatures or addition of alkalireagents as in the conventional process.

The electrochemical denaturation of DNA can be used in the amplificationof nucleic acids, e.g. in a polymerase chain reaction or ligase chainreaction amplification procedure. Thus the present invention provides aprocess for replicating a nucleic acid which comprises: separating thestrands of a sample double stranded nucleic acid in solution under theinfluence of an inorganic multivalent cation promoter and an electricalvoltage applied to the solution from an electrode; hybridising theseparated strands of the nucleic acid with at least one oligonucleotideprimer that hybridises with at least one of the strands of the denaturednucleic acid; synthesising an extension product of the or each primerwhich is sufficiently complementary to the respective strand of thenucleic acid to hybridise therewith; and separating the or eachextension product from the nucleic acid strand with which it ishybridised to obtain the extension product.

In such a polymerase mediated replication procedure, e.g. a polymerasechain reaction procedure, it may not be necessary in all cases to carryout denaturation to the point of producing wholly single-strandedmolecules of nucleic acid. It may be sufficient to produce a sufficientlocal and/or temporary weakening or separation of the double helix inthe primer hybridisation site to allow the primer to bind to its target.Once the primer is in position on a first of the target strands,rehybridisation of the target strands in the primer region will beprevented and the other target strands may be progressively displaced byextension of the primer or by further temporary weakening or separationprocesses.

Preferably, the said amplification process further comprises repeatingthe procedure defined above cyclicly, e.g. for more than 10 cycles, e.g.up to 20 or 30 cycles. In the amplification process the hybridisationstep is preferably carried out using two primers which are complementaryto different strands of the nucleic acid.

The denaturation to obtain the extension products as well as theoriginal denaturing of the target nucleic acid is preferably carried outby applying to the solution of the nucleic acid a voltage from anelectrode, the solution containing a promoter as described therein.

The process may be a standard or classical PCR process for amplifying atleast one specific nucleic acid sequence contained in a nucleic acid ora mixture of nucleic acids wherein each nucleic acid consists of twoseparate complementary strands, of equal or unequal length, whichprocess comprises:

(a) treating the strands with two oligonucleotide primers, for eachdifferent specific sequence being applied, under conditions such thatfor each different sequence being amplified an extension product of eachprimer is synthesised which is complementary to each nucleic acidstrand, wherein said primers are selected so as to be substantiallycomplementary to different strands of each specific sequence such thatthe extension product synthesised from one primer, when it is separatedfrom its complement, can serve as a template for synthesis of theextension produce of the other primer:

(b) separating the primer extension products from templates on whichthey were synthesised to produce single-stranded molecules in thepresence of a said promoter by applying a voltage from an electrode tothe reaction mixture: and

(c) treating the single-stranded molecules generated from step (b) withthe primers of step (a) under conditions such that a primer extensionproduct is synthesised using each of the single strands produced in step(b) as a template.

Alternatively, the process may be any variant of the classical orstandard PCR process, e.g. the so-called "inverted" or "inverse" PCRprocess or the "anchored" PCR process.

The invention therefore includes an amplification process as describedabove in which a primer is hybridised to a circular nucleic acid and isextended to form a duplex which is denatured by the denaturing processof the invention, the amplification process optionally being repeatedthrough one or more additional cycles.

More generally, the invention includes a process for amplifying a targetsequence of nucleic acid comprising hybridisation, amplification anddenaturation of nucleic acid (e.g. cycles of hybridising and denaturing)wherein said denaturation is produced by operating on a solutioncontaining said nucleic acid with an electrode in the presence of aninorganic multivalent cation promoter.

The process of the invention is applicable to the ligase chain reaction.Accordingly, the invention includes a process for amplifying a targetnucleic acid comprising the steps of:

(a) providing nucleic acid of a sample as single-stranded nucleic acid;

(b) providing in the sample at least four nucleic acid probes, wherein:i) the first and second of said probes are primary probes, and the thirdand fourth of said probes are secondary nucleic acid probes; ii) thefirst probe is a single strand capable of hybridising to a first segmentof a primary strand of the target nucleic acid; iii) the second probe isa single strand capable of hybridising to a second segment of saidprimary strand of the target nucleic acid; iv) the 5' end of the firstsegment of said primary strand of the target is positioned relative tothe 3' end of the second segment of said primary strand of the target toenable joining of the 3' end of the first probe to the 5' end of thesecond probe, when said probes are hybridised to said primary strand ofsaid target nucleic acid; v) the third probe is capable of hybridisingto the first probe; and iv) the fourth probe is capable of hybridisingto the second probe; and

(c) repeatedly or continuously: i) hybridising said probes with nucleicacid in said sample; ii) ligating hybridised probes to form reorganisedfused probe sequences; and iii) denaturing DNA in said sample byapplying a voltage from an electrode to the reaction mixture in thepresence of a said promoter.

In all of the amplification procedures described above the denaturationof the DNA to allow subsequent hybridisation with the primers can becarried out by the application of an appropriate potential to theelectrode. The process may be carried out stepwise involving successivecycles of denaturation or renaturation as in the existing thermalmethods of PCR and LCR, but it is also possible for it to be carried outcontinuously since the process of chain extension or ligation by theenzyme and subsequent strand separation by the electrochemical processcan continue in the same reaction as nucleic acid molecules insingle-stranded form will be free to hybridise with primers once theyleave the denaturing influence of the electrode. Thus, provided that theprimer will hybridise with the DNA an extension or ligation product willbe synthesised. The electrochemical DNA amplification technique can beused analytically to detect and analyse a very small sample of DNA eg asingle copy gene in an animal cell or a single cell of a bacterium.

The invention includes a kit for use in a process of detecting thepresence or absence of a predetermined nucleic acid sequence in a samplewhich kit comprises, an electrode, a counter electrode and optionally areference electrode, an oligonucleotide probe for said sequence and asource of an inorganic multivalent cation for use as a promoter inobtaining nucleic acid strand separation at said electrode. The probemay be labelled in any of the ways discussed above.

The invention also includes a kit for use in a process of nucleic acidamplification comprising an electrode, a counter electrode andoptionally a reference electrode, and a source of an inorganicmultivalent cation for use as a promoter in obtaining nucleic acidstrand separation at said electrode and at least one primer for use in aPCR procedure, or at least one primer for use in an LCR procedure,and/or a polymerase or a ligase, and/or nucleotides suitable for use ina PCR process.

Preferably, such kits includes a cell containing the electrodes.Preferably the kits include a suitable buffer for use in the detectionor amplification procedure.

BRIEF DESCRIPTION OF THE DRAWING

The invention will now be described with reference to the followingdrawings and examples.

FIG. 1 is a diagram of an electrochemical cell used for denaturation ofDNA.

In FIG. 1 there is shown a cell structure 10 comprising a workingcompartment 12 in which there is a body of DNA-containing solution, aworking electrode 14, a counter electrode 16, a FiVac seal 19, a Kwikfit adaptor 21 and a magnetic stirrer 18. A reference electrode 20 in aseparate side arm is connected via a "luggin" capillary 23 to thesolution in the working compartment 12. The working electrode, counterelectrode and reference electrode are connected together in apotentiostat arrangement so that a constant voltage is maintainedbetween the working electrode 14 and the reference electrode 20. Suchpotentiostat arrangements are well known (see for example "InstrumentalMethods in Electrochemistry" by The Southampton Electrochemistry Group,1985, John Wiley and Sons, p 19).

The electrode 14 is a circular glassy carbon rod of diameter 0.5 cm,narrowing to 0.25 cm at a height of 10 mM, and having an overall lengthof 9 cm inside a teflon sleeve of outside diameter 0.8 cm (supplied byOxford Electrodes, 18 Alexander Place, Abingdon, Oxon), and thereference electrode 16 is a 2 mm pin calomel (supplied by BDH No309/1030/02). The counter electrode is supported by a wire which issoldered to a brass sleeve 25 above the adaptor and passes down andexits the teflon sleeve 20 mm from the base of the working electrode.The wire attaches to a cylindrical platinum mesh counter-electrodesupplied by Oxford Electrodes which annularly surrounds the workingelectrode.

This cell is used in the following examples.

EXAMPLE 1

To the working chamber of the cell shown in FIG. 1 was added 900 μl ofdistilled water and 40 μg/ml of Calf Thymus DNA together with thepromoter shown in Table 1 below. The contents of the cell were subjectedto -1.0 V for up to 4 hours.

Samples were taken at 0, 30 mins, 1 hr, and 2 hrs. from commencement andanalysed on 1% agarose gel to observe the degree of denaturation.Results were as shown in Table 1.

    ______________________________________                                                                        Time for                                                          Promoter    complete                                      Run      Promoter   Concentration                                                                             denaturation                                  ______________________________________                                        1        Mg Cl.sub.2                                                                              10      mM    >4 hrs                                      2        Mg Cl.sub.2                                                                              30      mM    >4 hrs                                      3        Mg Cl.sub.2                                                                              100     mM    1-2 hours                                   Control  Methyl     30      mg/ml 1-2 hours                                            Viologen   (120    mM                                                                    approx)                                                   ______________________________________                                    

Thus it can be seen that in this system a minimum effective amount ofMg²⁺ as a promoter lies between 30 and 100 mM and that Mg²⁺ isapproximately as effective as a promoter as methyl viologen.

We claim:
 1. A process for denaturing double-stranded nucleic acidcomprising the steps of: applying a voltage to a solution containingsaid nucleic acid with an electrode; and converting at least aproportion of said nucleic acid to a wholly or partially single-strandedform, wherein the solution contains an effective concentration of amultivalent inorganic cation acting as a promotor which assists saiddenaturation.
 2. A process as claimed in claim 1, wherein a potential offrom -0.5 to -1.5 V is applied to said electrode with respect to saidsolution.
 3. A process as claimed in claim 2, wherein said voltage isfrom -0.8 to -1.1 V.
 4. A process as claimed in claim 1, wherein saidelectrode, a reference electrode and a counter-electrode are contactedwith said solution and a voltage is applied between said electrode andsaid counter-electrode so as to achieve a desired controlled voltagebetween said electrode and said reference electrode.
 5. A process asclaimed in claim 1, wherein the ionic strength of said solutionexcluding said promoter is no more than 250 mM.
 6. A process as claimedin claim 5, wherein the said ionic strength is no more than 100 mM.
 7. Aprocess as claimed in claim 5, wherein the said ionic strength is nomore than 50 mM.
 8. A process as claimed in claim 5, wherein the saidionic strength is no more than 25 mM.
 9. A process as claimed in claim5, wherein the said ionic strength is no more than 5 mM.
 10. A processas claimed in claim 1, wherein said promoter is magnesium ions.
 11. Aprocess as claimed in claim 1 wherein the concentration of said promotercation is from 1 mM to 250 mM.
 12. A process as claimed in claim 1,wherein the electrode is of carbon, gold or platinum.
 13. A process asclaimed claim 1, carried out at a temperature less than the meltingpoint of the double-stranded nucleic acid.
 14. A process as claimed inclaim 13, carried out at about ambient temperatures.
 15. A process asclaimed in claim 1 carried out at a pH of from 3 to
 10. 16. A process asclaimed in claim 15, carried out pH of about
 7. 17. A process as claimedin claim 1, wherein the nucleic acid is dissolved in an aqueous solutioncontaining a buffer whose nature and ionic strength are such as not tointerfere with strand separation of the nucleic acid.
 18. A process asclaimed in claim 1, wherein said nucleic acid is DNA.
 19. A process asclaimed in claim 1, wherein said nucleic acid comprises a DNA strand andan RNA strand.
 20. A process as claimed in claim 1, wherein said nucleicacid is a double stranded RNA.
 21. A process as claimed in claim 1,wherein said process is carried out as a denaturing step in a nucleicacid amplification procedure.
 22. A process for amplifying a targetsequence of nucleic acid comprising hybridisation replication anddenaturation of nucleic acid, wherein said denaturation is produced byapplying a voltage to a solution containing said nucleic acid with anelectrode wherein the solution contains an effective concentration of amultivalent inorganic cation acting as a promoter which assists saiddenaturation.
 23. A process as claimed in claim 22, which is apolymerase chain reaction amplification process or a ligase chainreaction amplification process.
 24. A process for replicating a nucleicacid which comprises: separating the strands of a sample double strandednucleic acid in solution to effect a denaturation under the influence ofan electrical voltage applied to the solution from an electrode;hybridising the separated strands of the nucleic acid with at least oneoligonucleotide primer that hybridises with at least one of the strandsof the denatured nucleic acid; synthesising an extension product of theor each primer which is sufficiently complementary to the respectivestrand of the nucleic acid to hybridise therewith; and separating the oreach extension product from the nucleic acid strand with which it ishybridised to obtain the extension product wherein the solution containsan effective concentration of a multivalent inorganic cation acting as apromoter which assists said denaturation.
 25. A process as claimed inclaim 24, which further involves repeating the procedure defined inclaim 24 cyclicly.
 26. A process as claimed in claim 24, wherein thehybridisation step is carried out using two primers which arecomplementary to different strands of the nucleic acid.
 27. A process asclaimed in claim 24, wherein the separating to obtain the extensionproduct is carried out by applying to a solution of the extensionproduct a voltage from an electrode.
 28. A process as claimed in claim24, for amplifying at least one specific nucleic acid sequence containedin a nucleic acid or a mixture of nucleic acids wherein each nucleicacid consists of two separate complementary strands, of equal or unequallength, which process comprises:(a) treating the strands with twooligonucleotide primers, for each different specific sequence beingamplified, under conditions such that for each different sequence beingamplified an extension product of each primer is synthesised which iscomplementary to each nucleic acid strand, wherein said primers areselected so as to be substantially complementary to different strands ofeach specific sequence such that the extension product synthesised fromone primer, when it is separated from its complement, can serve as atemplate for synthesis of the extention product of the other primer: (b)separating the primer extension products from the templates on whichthey were synthesised to produce single-stranded molecules by applying avoltage from an electrode to the reaction mixture wherein the reactionmixture contains an effective concentration of a multivalent inorganiccation acting as a promoter for said separation: and (c) treating thesingle-stranded molecules generated from step (b) with the primers ofstep (a) under conditions such that a primer extension product issynthesised using each of the single strands produced in step (b) as atemplate.
 29. A process as claimed in claim 23 for amplifying a targetnucleic acid comprising the steps of:(a) providing nucleic acid of asample as single-stranded nucleic acid: (b) providing in the sample atleast four nucleic acid probes, wherein: i) the first and second of saidprobes are primary probes, and the third and fourth of said probes aresecondary nucleic acid probes; ii) the first probe is a single strandcapable of hybridising to a first segment of a primary strand of thetarget nucleic acid; iii) the second probe is a single strand capable ofhybridising to a second segment of said primary strand of the targetnucleic acid; iv) the 5' end of the first segment of said primary strandof the target is positioned relative to the 3' end of the second segmentof said primary strand of the target to enable joining of the 3' end ofthe first probe to the 5' end of the second probe, when said probes arehybridised to said primary strand of said target nucleic acid: v) thethird probe is capable of hybridising to the first probe; and iv) thefourth probe is capable of hybridising to the second probe; and (c)repeatedly or continuously: i) hybridising said probes with nucleic acidin said sample; ii) ligating hybridised probes to form reorganised fusedprobe sequences; and iii) denaturing DNA in said sample by applying avoltage from an electrode to the reaction mixture, wherein the reactionmixture contains an effective concentration of a multivalent inorganiccation acting as a promoter for said separation.
 30. A process fordetecting the presence or absence of a predetermined nucleic acidsequence in a sample which comprises: denaturing a sampledouble-stranded nucleic acid by means of a voltage applied to the samplein a solution by means of an electrode, wherein the solution contains aneffective concentration of a multivalent inorganic cation acting as apromoter which assists said denaturation; hybridising the denaturednucleic acid with an oligonucleotide probe for the sequence; anddetermining whether the said hybridisation has occurred.